As a new pollutant, microplastics have been widely distributed in Marine and coastal ecosystems, posing a serious threat to ecological environment. As an important ecological barrier, the microplastic pollution problem of mangroves is particularly prominent. The deposition of microplastics from the ocean and land into mangrove forests affects the physiological pathways of mangroves, impedes the growth and development of mangroves, and has an ecological and economic impact on the local area, so we intend to use synthetic biology to degrade microplastics from the soil surface to the deep soil. In order to degrade plastics, we used Pseudomonas aeruginosa. According to the four steps of microplastic depolymerization - degradation - assimilation - mineralization, we designed several modules of adsorption degradation/enhanced assimilation/enhanced mineralization, which greatly enhanced the ability of Pseudomonas aeruginosa to degrade microplastics. At present, how to degrade microplastics in deep soil under anaerobic conditions is also a difficult problem to solve. At the same time, we specially introduced the bacterial hemoglobin gene vgb to help Pseudomonas aeruginosa to degrade microplastics under anaerobic conditions. At the same time, microplastics will release organic carbon deposited in mangroves and destroy mangroves as an important carbon sink. It is known that Rhodopseudomonas can sequester carbon when there is no light in the deep soil and electron donors are available. Therefore, in order to enhance electron transfer, we introduced nqrF and Papila1-61M3 genes into Pseudomonas aeruginosa. At the same time, we introduced bscA and bscB gene into Rhodopseudomonas palustris, converts carbon dioxide into cellulose for carbon sequestration, and introduced nadK, nadM, pntA, pntB which increases NADPH production to speed up the BBC cycle.
We chose Pseudomonas aeruginosa PAO1 as the chassis. Pseudomonas aeruginosa is a common bacterium in mangrove ecosystems, so the introduction of engineered bacteria will not disrupt the original ecology of mangrove forests, ensuring its effectiveness. Pseudomonas aeruginosa is able to utilise a wide range of carbon substrates as a source of carbon and energy. It thrives over a wide range of temperatures, is ubiquitous and has the potential to form biofilms in the environment, thereby enhancing the degradation of polyethylene microplastics.
Rhodopseudomonas palustris CGA009, a widely studied and used biological chassis, is a bacterium with the properties of extraordinary metabolic versatility, carbon source diversity and metabolite diversity. It indicates that R. palustris CGA009 can use CO2 as the carbon source, which is fixed by the Calvin Bassham Benson (CBB) cycle to participate in cell growth metabolism. Furthermore, many researches indicates the potential of R. palustris as a chassis organism for biopolymers and their building blocks production, such as polysaccharide. In addition, after an electrosyntrophic coculture is formed, R. palustris CGA009 can grow autotrophically under dark, anoxic conditions through syntrophic interspecies electron transfer.
Biodegradation is mainly divided into 4 steps: (1) Bio-deterioration: microbial communities and abiotic factors work together to cut polymers into fragments; (2) Depolymerization: microorganisms secrete enzymes and free radicals to convert polymers into oligomers, dimers or monomers; (3) Assimilation: the depolymerized molecules are recognized by the receptors on the surface of the microorganism and cross the plasma membrane into the microbial cell; (4) Mineralization: The depolymerized molecules are metabolized and oxidized into small molecule compounds such as CO2、N2、CH4 and H2O in the microbial cells. In our project the PE degradation ability of Pseudomonas aeruginosa will be improved according to the above four steps.
We engineered two parts of the Passenger protein: the PE-binding peptide and the PE enzyme. We used modelling to predict the sequence of the PE-binding peptide, whose hydrophobic and van der Waals forces help our engineered bacteria bind to the microplastic surface and enhance the ability of Pseudomonas aeruginosa to aggregate near the microplastic. The most limited step in the plastic degradation process is actually the degradation of microplastics from polymers to monomers, so we fused an enzyme capable of depolymerising PE downstream of PEBP. Tianjin University has verified that this enzyme can be expressed in Escherichia coli and play a good role in extracellular environment last year, so we chose this enzyme as our PE degrading enzyme.We chose PEase (from Tianjin University), an enzyme found in the saliva of waxworms that oxidises PE into polyethylene plastic.
Fig.1 Passenger Protein Schematic
To immobilise these two proteins on the bacterial surface. We chose autotransporters as the anchoring method and predicted the signal peptide and β-barrel region of the estA protein of PAO1. As part of the transporter, they bind to both ends of our Passenger protein so that our protein is well anchored to the bacterial outer membrane. In addition, we added a linker, tags, the enzyme cutting sites and other components in the middle of these modules to facilitate the expression, purification and transformation of our components.
We construct AlkB2-Rd45-Adh fusion protein. Membrane-bounding Alkane hydroxylase (AlkB2) can hydroxylate straight alkanes to alcohols and transport it into the cytoplasm, which improves the ability of Pseudomonas aeruginosa to assimilate alkanes and degrade PE plastics. As AlkB2 needs one or more redox partners to obtain electrons for monooxygenation reactions, we link rubredoxin Rb45 from alkane hydroxylase-rubredoxin fusion gene alkW1 in Dietzia sp. DQ12-45-1b after AlkB211. This protein enables AlkB2 to play a better role without coupling more rubredoxin proteins.
The pathway that now has been proposed for the degradation of n-alkane includes initial hydroxylation at the terminal ω carbon by a hydroxylase like AlkB2 or CYP, further followed by the formation of the corresponding ketone by a primary alcohol dehydrogenase (Adh). Besides. Studies have shown that AlkB will consume NADH while Adh will consume NAD+ and produce NADH in catalyzation. In order to facilitate the further degradation of n-alkane and reproduction of NADH, we fuse AdhA after the Rd45 protein.
AlkB along with Rd45 and Adh construct a rather complete assimilation and mineralization module. However, AlkB is known for hydroxylate terminal or subterminal carbon on long-chain and medium-chain alkane but not short-chain alkane. To make up for the insufficient hydroxylation ability of AlkB to short-chain alkane and the inability to conduct in-chain hydroxylase. Therefore, we also introduced P450camY96F enzyme that can degrade short-chain alkane and conduct in-chain hydroxylate into Pseudomonas aeruginosa. It has been proved that this enzyme can greatly accelerate the degradation efficiency of PE in the case of heterologous expression.（待放图3）
Studies have shown that microplastics in mangrove soil sediments are vertically distributed at a depth of 0-100cm, and our chassis are aerobic symbiotic bacteria attached to the roots of mangrove plants, and oxygen is required for the degradation of PE, so the degradation of microplastics in deep soil and anaerobic areas that may be caused by tides faces problems.
VHb is a single-domain hemoglobin (SDHb) which can efficiently bind oxygen and transport it to the respiratory chain by interacting with terminal oxidases, especially under oxygen-restricted conditions. In this way, VHb significantly enhances the regeneration of NAD+, which is one of the substrates of most of our monooxygenase. In addition, VHb can also interact with transcriptional regulators responsible for oxygen responses, triggering oxidative phosphorylation in cell. （待放图4）
In this regard, the transfer of the vgb gene of the bacterial oxygen-carrying protein VHb enabled: enhancement of the enzymatic activity of the polyethylene mineralisation pathway of the engineering bacteria to accelerate microplastic degradation; and endowment of the engineering bacteria with anaerobic environmental resilience to ensure that they can degrade plastics in anaerobic zones deep in the soil. Ultimately, full coverage of the breadth and depth of plastic degradation by engineering bacteria in mangroves will be achieved.
Module 4-5 :
Pseudomonas aeruginosa, a thriving microbe in bioelectrochemical systems (BES), produces versatile phenazine redox mediators. BES, especially microbial fuel cells (MFCs), are rapidly developing for renewable energy and bioremediation. MFCs generate electricity via extracellular electron transfer from microbes degrading organic matter.
The most commonly described transfer mechanisms are direct electron transfer via direct cell contact or protein nanowires and mediated electron transfer via secondary or primary metabolites.
Attempts to improve the biological efficiency of MFCs have therefore focused on understanding and improving these mechanisms. In mediated electron transfer, microorganisms utilize endogenous or exogenous soluble redox mediators that enable transmission of electrons to an external electron acceptor. In bacteria, endogenous secondary metabolites used as mediators include riboflavins in Shewanella, phenazines in Pseudomonas, and quinones in Lactococcus. These molecules undergo reversible oxidation and reduction and hence can be used repeatedly as electron shuttles.
In mixed microbial communities and biofilms, redox mediators may be shared among different species, potentially fostering syntrophic interactions. Naturally, microbial consortia are formed within these communities, characterized by complex interactions that often enhance resource utilization. Further research has revealed synergistic relationships involving both native redox mediators and non-redox mediator producers, with these effects being especially significant under oxygen-limited conditions. P. aeruginosa-derived redox mediators have been demonstrated to facilitate extracellular electron transfer in a synergistic interaction with other strains. This provides a good basis for the linkage design with Rhodopseudomonas palustris in the deep soil in our project.
So one of the key challenges in enhancing the performance of extracellular electron transfer (EET), particularly for the strain Pseudomonas aeruginosa PAO1, is improving the efficiency of microbial electron transfer to the anode and The number of microbial nanowires.
Module 6 :
We degraded PE microplastics in mangrove soils using engineered
PAO1
. However, microplastics and their biodegradation release
CO2, altering water nutrients, decreasing microbes, and
impacting carbon-nitrogen cycles.
We employed Rhodopseudomonas CGA009, abundant in mangroves, as an auxiliary to absorb CO2 from PE degradation by PAO1. Leveraging its CO2 uptake, we modified CGA009 to optimize carbon absorption, minimizing emissions during degradation.
We will construct a truncated version of the PaPilA gene containing only the first 61 amino acids of the N-terminus, and perform a PaPilA1–61M3 plasmid with three amino acid mutations (E32Y, L51F, and G57Y) to greatly increase the biopotential output of PAO1.
Fig.1 Passenger Protein Schematic
Researches show that phenazine reductase activity in membrane fractions is attributable to the three NADH dehydrogenases present in P. aeruginosa and that their order of phenazine reductase activity is Nqr > Nuo > Ndh(15). Besides, the Nqr complex plays a role in the extracellular electron transport (EET) process of Pseudomonas aeruginosa, particularly under anaerobic conditions, by promoting the reduction of phenazines to facilitate energy production and the establishment of proton motive force.
Therefore, by increasing the activity of Nqr, the electron transport capacity of PAO1 can be improved.
Fig.1 Passenger Protein Schematic
CO2 is slightly soluble in water at room temperature (about 600:1) and produces mainly carbonic acid in water, but also small amounts of carbonate and bicarbonate. Therefore, carbon dioxide will be released from the water body under heating and shocking, so the carbon dioxide released by engineered bacteria in mangrove environments will escape from the water volume easily; in addition, during the process of degradation of Pseudomonas aeruginosa and Rhodopseudomonas palustris will form biofilms on the surface of microplastics, which CO2 transfer will be severely hindered. Therefore, we need to transform CO2 into a kind of substance that can be easily delivered in biofilms.
Dissolved inorganic carbon (DIC) toolkit is a system composed by inorganic transporter and carbonic anhydrase (CA) in autotrophs using Calvin Benson Basel cycle (CBB cycle). We create the DIC toolkit in Rhodopseudomonas palustris CGA009 and the delivery relationship between the two engineered bacteria.
Fig.1 The schema of the DIC toolkit in the engineered bacteria and interactions
Pseudomonas aeruginosa PAO1 can encode three cytoplasmic β-CAs: psCA1, psCA2, and psCA3. CAs can catalyze the reversible conversion of CO2 to bicarbonate: $$CO_2 + H_2O ⇌ HCO_3^- + H^+$$
Among them, psCA1 (encoded by the gene PAO102 or psCA1) is a β-carbonic anhydrase of type I, which has the highest conversion efficiency among the three and its secondary structure is not affected by pH.
It's known that there is the Bicarbonate transporter BicA (from Bicarbonate transporter BicA [Pseudomonas aeruginosa] - Protein - NCBI (nih.gov) ) on the membrane of Pseudomonas aeruginosa PAO1. We take advantage of psCA1 to transform CO2 that is produced by Pseudomonas aeruginosa PAO1 during the process of PE degradation to HCO3-, and later transport HCO3- out of the cell via BicA.
Rhodopseudomonas palustris CGA009 is a type of gram-negative bacteria that contain two cellular compartments (cytoplasm and periplasm). An α-CAs (encoded by acaP) is present in its periplasm. It has been shown that this kind of CA is inactive in aerobic environment and active in anoxic environment. Microplastics in mangroves are distributed in low-oxygen environments such as water and soil. In Rhodopseudomonas palustris CGA009, the function of this kind of CA is to accelerate the speed of conversion from HCO3- to CO2 when the inorganic carbon source is bicarbonate, and fix CO2 by ribulose-bisphosphate carboxylase-oxygenase (RubisCo), finally conversing to utilizable organic matter.
Fig.2 the schematic of the CBB cycle
There is bicarbonate membrane transporter named ABC transporter substrate-binding protein existing in the cell membrane of Rhodopseudomonas palustris CGA009 (from ABC transporter substrate-binding protein [Rhodopseudomonas palustris] - Protein - NCBI (nih.gov) ). After HCO3- taken in engineered bacteria, α-CAs catalyzes the process of HCO3-/ CO2 conversion to produce CO2 for CBB cycle after caught by RubisCo.
In order to process as much CO2 as possible from the decomposition of microplastics, we need to increase the efficiency of engineered bacteria in HCO3-/CO2 conversion. Given that the structures of all three CAs in Pseudomonas aeruginosa PAO1 are highly conserved and adjusting the structure can easily lead to inactivation. Accordingly, we apply the method of psCA1 multiple copies to produce bicarbonate more efficiently.
For the same reason, in order to enhance the efficiency of the conversion from HCO3- to CO2, we still take the approach of multiple copies of acaP.
In addition to some parts of bicarbonate delivered to Rhodopseudomonas palustris CGA009, some of them also combines with Ca2+ inside and outside the engineered Pseudomonas aeruginosa PAO1 to form CaCO3 deposits. However, it has been shown that: (1) Calcium carbonate deposition is easy to form when the water body is in calm and a high concentration of CO2 exists in it, but little calcium deposition occurs when the water is in waving and in a common CO2 conditions; (2) If calcium carbonate deposition is produced, it also can contribute to the stabilization organic matter and reduces the amount of carbon dioxide released from soil. (3) And even the small amount of calcium carbonate is produced, it is also important in the formation of biofilm. Therefore, the formation of calcium carbonate deposits is not considered as an issue in our project.
Rhodopseudomonas palustris is renowned for its ability to function in the four known metabolisms of life. This unique ability allows R. palustris to convert light or electrical energy into chemical energy in organic matter. It suggests that R. palustris can drive the fixation of carbon dioxide and the synthesis of bacterial cellulose through syntrophic interspecies electron transfer.
As a precursor of bacterial cellulose, the synthesis of glucose requires glyceraldehyde-3-phosphate. However, the intracellular level of NADPH limits the efficiency of the Calvin cycle of R. palustris in producing glyceraldehyde-3-phosphate. Since the Calvin cycle is constrained by the concentration of NADPH and NADP+, the synthetic biological strategy to improve the intracellular NADPH and NADP+ level is of great value to prompt the efficiency of Calvin cycle.
The lack of NAD+ may have an adverse effect on Rhodopseudomonas palustris CGA009. Nicotinamide mononucleotide (NMN) is an important precursor of NAD+, and Rhodopseudomonas palustris CGA009 cannot directly synthesize NAD+ from NMN, and the synthesis efficiency of NAD+ is low. Therefore, we introduced the gene encoding nicotinamide-nucleotide adenylyltransferase NadM of nadK from Francisella tularensis SCHU S4 to convert NMN directly to NAD+ and avoid depletion of NAD+.
Because phosphorylation of NAD by NAD kinase (NADK) is the only known mechanism by which NADP is produced de novo3, in order to increase NADP+ synthesis, we introduced the gene encoding NADK of nadK from Rhodopseudomonas palustris RCB100 to increase NADP+ synthesis.
In addition to the above two modules, we introduced the gene encoding PntAB of pntA and pntB from Escherichia coli MG1655 to increase the original rate of the conversion of NADP+ to NADPH. Ultimately, since the biosynthesis of glyceraldehyde-3-phosphate depends on the intracellular level of NADPH, module 1-3 will promote the production of glucose.
In the microbial carbon sequestration pathway, carbon is usually accumulated through refractory substances in biological residues, so increasing the synthesis and accumulation of cellulose of refractory substances can effectively retain carbon in the soil after bacterial death.
Cellulose synthesis usually involves multiple subunits, of which BcsA and BcsB are the core catalytic subunits responsible for the synthesis of cellulose chains. Therefore, we increased cellulose synthesis by transferring to bcsa and bcsb derived from Pseudomonas pactida NBRC 14164.
